Dear Patrick,
it depends on how strongly your protein binds. In fact for a
protein I used to work with I would purposefully load a sample in
phosphate (I forget how much, maybe 100mM) so that the binding in my
desired protein was improved. This was after a lot of preliminary work
of course. What is the pI of your protein? Have you tried small
scale experiments? Mike.
On 9 May 2001, Patrick Lynch wrote:
> Hi all,
>> I'm going to carry out anion exchange chromatography on eluted fractions
> from a Histidine binding column. Normally, I just dilute with Tris buffer
> (to reduce the 0.5M NaCl from the His binding column) and apply to the
> Mono-Q column. Question! There's 20 mM phosphate in the eluted fraction as
> well - how much should this be diluted for application to the Mono-Q as you
> are not supposed to use negatively charged buffer ions with anion exchange?
>> Thanks,
>> Patrick
>> PS I've tried using a buffer exchange column but the protein of interest
> didn't like it and anyway, it's another step!
>>>> ---
>